RNA interference (RNAi) is an evolutionarily conserved process in eukaryotic organisms that serves as a regulatory mechanism to target specific RNAs for degradation. A hallmark of RNAi is the incorporation of short interfering RNAs (siRNAs) into RISC (RNA-induced silencing complex). RISC or related complexes are also thought to be involved as defense systems against virus infections, but direct biochemical proof remains to be provided for any organism. In the Preliminary Studies section, evidence is presented for the isolation of an in vitro active Tomato bushy stunt virus (TBSV)-activated siRNA-protein complex with bona fide RISC properties and with novel features not previously reported that provide an explanation for several biological observations associated with infection. The newly developed system and the findings provide a unique opportunity to study the properties of antiviral RISC in detail to reveal principles that may apply to RISC of other systems and thus can potentially yield information to design improved strategies for RNAi-based therapies. This fits the mission of the agency to pursue "fundamental knowledge about the nature and behavior of living systems and the application of that knowledge." The long-term goal is to apply TBSV as a model system for studying the biochemical properties of RISC for future comparison of antiviral RISC-like complexes in other organisms. Within this scope, the objective of this RO3 application is to characterize the anti-TBSV RISC in detail using techniques specifically adapted for this purpose in the laboratory, and to compare the properties with RISC induced by other plant viruses. The underlying hypothesis of the application is that antiviral RISCs not only share many properties but also have biochemical attributes that are virus- specific. To test this, the specific aims of this application are to: 1) Characterize RISC from TBSV infected plants, and 2) Characterize RISC isolated from plants infected with viruses other than TBSV. Relevance for Public Health. First, a better understanding of how organisms defend themselves against certain viruses will be beneficial towards our understanding and implementation of antiviral therapies. Second, since virus infections produce high amounts of small RNAs (i.e. siRNAs); they represent useful model systems towards improving new strategies for therapeutic administration of high dosages of siRNAs. [unreadable] [unreadable]